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Lecture 12-30 Transcription Translation Transcription 1 Transcription Translation Translation Protein Synthesis Part 1: Take Home Message Required reading: Stryer Ch. 34, p. 875-888 Ch. 33 p. 849-850 1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid AA (codons). 2) The mRNA message is read by tRNA through the use of a three base complement to the three 3 base word (anticodon). 3) A specific amino acid AA is conjugated to a specific tRNA. 4) Amino acid AA side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to conjugate a specific three 3 base message with a specific amino acid AA. Lecture 12-30 Transcription Translation 2 Table 1. The genetic code. for mRNA 1st position (5' end) U C A G Genetic Code 2nd position 3rd position (3' end)--> U C A G Phe Phe Leu Leu Ser Ser Ser Ser Tyr Tyr STOP STOP Cys Cys STOP SelenoCys Trp Mitochondria Trp Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg Ile Ile Ile Met init Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G U C A G U C A G U C A G Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into valine and glutamic acid, respectively. Note that those codons with specify the more hydrophobic amino acids AA. U or C as the second 2 nucleotide tend to Lecture 12-30 Transcription Translation 3 Structure of Transfer RNA Amino Aci d O H Conjugati on A 3' Site C 5' P C O H +H C N H O C Du pl ex A 3' R C regi on C DHU l oop T C l oop C A G T C G G O Du pl ex regi on H H2C H2C N N O O HN di-hydro-uri dine (DHU) NH N O ps eu do-uri dine ( ) U Di-hydro-uridine (DHU) Anti Codon Pseudo-uridine () Classes of Aminoacyl-tRNA Synthetases • Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger Amino Acids) • Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr (Generally the smaller amino acids) H H tRNA N 1 N 5 O 6 N 7 4 2 O P O 5' 8N N H O 3 9 O H 4' H Charged tRNA -76 -76 1' H 3' H O C O C H 2' H H tRNA Activation by aminoacyl tRNA synthases 1. Amino-acyl-AMP formation + N H H Lecture 12-30 Transcription Translation H O H O O O R P P O O O O H O O + C O O P P Adenine + O H N C O R O H P O O H H O + O H H Adenine O + C O O O P H N H H C O H H O O O H H H O O O H H O + PO OH H O H H O O P H H OH O H O 4 2. Aminoacyl transfer to the appropriate tRNA + AMP1- R H H H O + O N CC H O O H O Adenine O P O H H H O H + O ACC OH Adenine O P t RNA R H H O + N CC H H O O O + O ACC t RNA H H H O H H OH Lecture 12-30 Transcription Translation 5 tRNA Recognition by Synthetases • Specific recognition of the anticodon; ex. an change the anticodon for tRNATrp to that for methionine and get good acylation by tRNAMet. •Stem sequences can be crucial; ex. tRNAAla depends on GC at position 3:70 doesn't care what the codon is. • Both the stem regions and anticodon are needed; ex. tRNA Gln. tRNA Recognition by Synthetases tRNA-Ala tRNA-Phe Trna- Ser OH A 3' C C OH A 3' 5' P C C 5' P OH A C C 5' P 3' U70 G3 C T C G A C T C G A GG GG A C11 C T C G GG G24 U U G34 A35 A36 U tRNA Recognition by Synthetases S tem and ATP S ite t-RNA Anticodon Recognition Additional Recognition Lecture 12-30 Transcription Translation 6 tRNA Synthetase Proofreading Ile Large Smaller Acylation Site Hydrolytic Site Large Smaller Acylation Site Hydrolytic Site H H H C H H HC H H C H H + C O O H N H H C HH C N C C Ile t RNA O t RNA Difference Ile +H H H O H H C H H H C H H + H N C H O C C O C H H H H H C C O t RNA Correct Acylation Isol eucine Isol eucine H H + N H t RNA O H Mis-acylation tRNA Synthetase Proofreading Val Hydrophobic Polar Acylation Site Hydrolytic Site H H H C H H H N C H H H H O + O H H + N H V al t RNA O H H H C O H Difference t RNA H H CH C H V al O H H C H O H H H H Hydrophobic Polar Acylation Site Hydrolytic Site O + O N H O Correct Acylation H + N H H t RNA Valine Valine t RNA O Mis-acylation Lecture 12-30 Transcription Translation 7 How do we go from mRNA to Protein? Incoming tRNA Table 1. The genetic code. for mRNA 1st position (5' end) U C A G Genetic Code 2nd position 3rd position (3' end)--> U C A G Phe Phe Leu Leu Ser Ser Ser Ser Tyr Tyr STOP STOP Cys Cys STOP SelenoCys Trp Mitochondria Trp Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg Ile Ile Ile Met init Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G U C A G U C A G U C A G Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into valine and glutamic acid, respectively. Note that those codons with specify the more hydrophobic amino acids AA. Codon : Anticodon First 1st Base of Anticodon is Variable 3 2 1 t RNA- 3'-X Y Z- -5' anticodon mRNA- 5'-XΥYΥ ZΥ -3' codon 1 2 3 Third 3rd Base of Codon is Variable U or C as the second 2 nucleotide tend to Example tRNAPhe 3Υ--A--A--GMe -5Υ 5Υ-U - U - C -3Υ 3Υ--A--A--GMe -5Υ 5Υ-U - U - U -3Υ Lecture 12-30 Transcription Translation 8 Wobble pairing rules 5’ anticodon base 3’ codon base C G A U U A or G G C or U U , C, or A I tRNA Anticodon-Codon Recognition Anticodon 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ U – 3’ 5’ – C – G – C – 3’ 5’ – C – G – A – 3’ Anticodon 3’ – C – G – G – 5’ 3’ – C – G – G – 5’ U – 3’ 5’ – C – G – C – 3’ Anticodon 3’ – C – G – U – 5’ Codon 5’ – C – G – A – 3’ 3’ – C – G – U – 5’ 5’ – C – G – G – 3’ Anticodon 3’ – C – G – C – 5’ 3’ – C – G – A – 5’ Codon 5’ – C – G – Codon 5’ – C – G – Codon 5’ – C – G – 5’ – C – G – G – 3’ U – 3’ tRNA Anticodon-Codon Recognition Adenosine H H N N N Inosine Guanosine O O H N N N N H N N N Ribose N H N N N H H Anticodon 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ 3’ – C – G – I – 5’ Codon 5’ – C – G – A – 3’ 5’ – C – G – N N ¥C 1 N 5’ – C – G – C – 3’ O HN H NH N N O ¥C 1 ¥C1 N I------C base pair N N O HN N N NH N N U – 3’ O H H N O N H N H ¥C1 I-----A base pair NH O N ¥C1 ¥C1 N I----- U base pair Nonsense suppression Nonsense mutations = change of codon for an AA to S TOP Usually lethal Πtruncated protein Can be rescued by mutation in a different part of the genome Mechanism: tRNA gene mutation Example: E. Coli Amber suppressor tRNATyr anticodon change G U A C U A Mutated tRNA recognized S top codon as Tyr and prevents chain termination Lecture 12-30 Transcription Translation 9 Table 1. The genetic code. for mRNA 1st position (5' end) U C A G Genetic Code 2nd position 3rd position (3' end)--> U C A G Phe Phe Leu Leu Ser Ser Ser Ser Tyr Tyr STOP STOP Cys Cys STOP SelenoCys Trp Mitochondria Trp Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg Ile Ile Ile Met init Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G U C A G U C A G U C A G Sets of three 3 nucleotides (codons) in an mRNA molecule are translated into amino acids AA in the course of protein synthesis according to the rules shown. The codons G U G and GAG, for example, are translated into valine and glutamic acid, respectively. Note that those codons with specify the more hydrophobic amino acids AA. U or C as the second 2 nucleotide tend to Protein Synthesis Part 2: Take Home Message 1) Translation always proceeds by reading the mRNA sequence from the 5’ to the 3’ end. 2) Amino acids are assembled in to proteins with the first amino acid having a free amino group —N 3 + (N-terminus) and the last having a free carboxylate—COO— (C-terminus). 3) Synthesis of the protein is processive and contains steps; a) initiation, b) elongation and c) termination Lecture 12-30 Transcription Translation Overall Ribosome Structure: 10 Translation Machine 23S RNA 50S S ubunit 34 proteins 5S RNA 16 S RNA 21 proteins 30S S ubunit 30S S ubunit 70S Ribosome Large subunit 50 S ¥ 70S can be dissociated into two 2 subunits: 50S and 30 S ¥ S mall subunit (30S ) contains 16S RNA and 21 proteins ¥ Large subunit (50S ) contains 23S RNA, 5S RNA, and 34 proteins ¥ 50S and 30S can come back together to form 70S ¥ rRNA is folded and has distinct tertiary 3΅ structure ¥ rRNAs probably dominate the ribosome function, 23S necessary for catalytic function S mall subunit 30 S • tRNA site, peptidyl transferase, GTPase and Exit site 50S S ubunit Lecture 12-30 Transcription Translation 11 T T --GA T -- T C T -AAA-- T AA- 3’ Translating the Message DNA 5'-ATG---GCC- T RNA 5' -A U G--GCC- U U U --GA U -- U C U -AAA-- U AA--3’ Protein N- Met 5' Elongation Ala Phe Asp Ser Overview of Translation Lys Stop -C DNA RNA Pol { Stop Protein 50S Ri bosomes 3' 30S ¥ M ultiple ribosomes can be involved simultaneously, max. density one 1 ribosome per 80 nucleotides ¥ All mRNAs contain signals for starting and ending polypeptide synthesis. 70S mRNA 5' Initiation Protein Synthesis Part 1: Take Home Message 1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid. 2) The mRNA message is read by tRNA through the use of a three base complement to the three 3 base word. 3) A specific amino acid is conjugated to a specific tRNA (three base word). 4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to conjugate a specific three base message with a specific amino acid AA.